Prothymosin alpha is a small, highly acidic protein originally thought to be a precursor for secreted thymic peptides. Our studies are not consistent with this view; they indicate that prothymosin alpha is regulated in a cell cycle dependent manner, and that cell division depends on sufficient quantities of prothymosin alpha. These results are based, in part, on a two step purification of prothymosin alpha which produces a virtually homogeneous protein. Methods have also been devised for localizing prothymosin alpha in the cell. Our results indicate that the protein is able to enter the nucleus and that a carboxyl terminal sequence is both necessary and sufficient for this process. The mechanism of action of the ribonuclease H activity of reverse transcriptase from avian myeloblastosis virus and human immunodeficiency virus has been investigated using carefully constructed substrates which eliminate enzyme processivity. Under these conditions, the time course of the reaction is biphasic with a burst preceding a much slower steady state rate. The existence of a burst made possible the evaluation of three out of four rate constants that govern the hydrolysis of RNA; the rate limiting step is the regeneration of free enzyme. From the kinetics of the burst and the steady state rate, the values of KD and KM for the DNA:RNA hybrid in the RNase H reaction were determined and compared with the value of KM for the hybrid in the polymerase reaction. Further insight into substrate binding was obtained using inhibitors. The inhibition by ribonucleoside vanadyl complexes indicated that both the value of Ki for the inhibitor and the complex pattern of inhibition were the same for the DNA:RNA hybrid regardless of which reaction was being studied. The combined data fit a model in which the DNA:RNA substrate is rigidly held in a single substrate binding site formed by the interaction of the two subunits of HIV reverse transcriptase. The binding site for deoxyribonucleoside triphosphates is separate and not affected by vanadyl complexes.